【作者】 王道红；

【导师】 王伯初；

【作者基本信息】 重庆大学 ， 生物医学工程， 2004， 硕士

【摘要】 研究表明，一些环境胁迫如干旱、水、盐、电场和机械刺激等能够诱导植物基因特异性表达。然而，作为自然界最为常见的一种环境胁迫——声音，其对植物基因表达影响的研究却不深入。有研究表明，声波作用不会导致植物遗传物质发生变异，而是在转录水平上造成基因表达方式发生改变。但声波作用后是植物的哪段基因片段响应声波刺激而表达方式变化还未见报道。本文以菊花作为实验材料，对声波处理后菊花基因的差异表达做初步研究。 本文首先提取了菊花总RNA。用1000HZ和100dB的声波处理经二次传代的菊花幼苗9天，每天60分钟。分别提取了处理组和对照组菊花的总RNA；结果表明，提取的总RNA浓度、纯度和完整性均很好，能够满足后续实验的要求；且声波处理组菊花总RNA得率要大于对照组(见表4．1)。 首先以提取的刺激组和对照组总RNA为模板，运用mRNA逆转录差异显示技术(DDRT-PCR)筛选受声波诱导的差异表达基因片段。在变性聚丙烯酰胺凝胶上分离到8个差异片段(见图5．7，5．8)，分别为SA5-1、SA5-2、SA7、CA7、SC1、SC8-1、SC8-2、CG1；对上述8个差异片段重扩增后在琼脂糖凝胶上显示的结果表明，8个片段中只有SA5-2、SC1和SC8-1(见图5．9)三个片段分子量和变性聚丙烯酰胺凝胶上显示的条带分子量一致。 为了进一步确定SA5-2、SC1和SC8-1是否为真正受声波诱导而表达的条带，对其进行了Northern点杂交。结果表明，SA5-2和SC8-1与声波处理组菊花总RNA杂交后在胶片上清晰显影，而SC1则不显影(见图5．10，5．11，5．12)。说明SC1为假阳性，而SA5-2和SC8-1为阳性条带。其中SC8-1是受到声波处理后而特异性表达的，而SA5-2是优势表达条带。其分子量分别约为270bp和290bp。可以进一步对其克隆，分析序列和功能描述。 分析菊花和拟南芥经声波处理后产生的差异条带。经比较发现，菊花中的特异片段SA5-2和拟南芥中的特异片段SA3(见图5．13，5．14)有相似之处：扩增这两条条带的引物组合相近，锚定引物均为AAG CTT TTT TTT TTA，随机引物分别为AAGCTTAGTAGGC和AAGCTTTGGTCGA；分子量也接近，分别约为290bp和270bp，可以对其作进一步对比分析。更多还原

【Abstract】 Much work has been done about some environment stresses such as drought, water flow, salinity and mechanical stimulation inducing the differential expression of genes in plant. However, Reports about the study of the effects of the sound, a widespread environment stress, on the gene expression of plants are rare. Only the preliminary study showed that heredity substance had no variation under sound stimulation, but gene expression was altered. However, it is still unclear which gene fragments in the plant are differentially expressed under sound stimulation. In this thesis, chrysanthemum is chosen as. test material and the differential expression of chrysanthemum genes are preliminarily studied to screen out genes induced by the sound wave.Firstly, the total RNA of chrysanthemum is extracted. The chrysanthemum seedling is treated by sound wave with a certain intensity (100dB) and frequency (1000 Hz) for 60 minutes a day, After 9 days, the total RNA of the treated group and the control group are extracted with RNase Plant Mini kit. The results show that high-quality total RNA has been obtained. The concentration, the purity and the integrality of the total RNA can satisfy the need of the subsequent tests. Moreover, the yield rate of total RNA is larger in the treated group than in the control group(Tab 4.1).As a template, the total RNA extracted from the treated group and the control group is reversely transcribed into cDNA. mRNA reverse transcription differential display PCR(DDRT-PCR) is applied to screen out the differential expression gene fragments induced by the sound wave. 8 pieces of bands, which are SA5-1, SA5-2, SA7, CA7, SCI, SC8-1, SC8-2, CGI respectively, are isolated by the polypropylene acyl gel electrophoresis(Fig 5.7, 5.8). After being re-amplified, only the 3 bands, which are SA5-2, SCI and SC8-1, appear on the agarose gel(Fig 5.9). The molecular weight of the three gene fragments on the agarose gel and on the polypropylene acyl gel is approximate. They are the fragments that are looked for.Northern dot hybridization is used to further confirm whether SA5-2, SCI and SC8-1 are the differential expression gene induced by the sound wave or not. The results demonstrate that SA5-2 and SC8-1 are really the differential expression genes, whose molecular weight are 290bp and 270bp respectively. Thereinto, SA5-2 is dominantly expressed, and SC8-1 is differentially expressed, while SCI is fake positivefragment(Fig 5.10,5.11,5.12). SA5-2 and SC8-1 are goning to be cloned and sequenced, and then their functions further analyzed.By analyzing the differential gene fragments in the chrysanthemum and in the Arabidopsis thaliana, it is found that SA5-2 in the chrysanthemum and SA3 in the Arabidopsis thaliana are very similar(Fig 5.13,5.14), which are amplified from primer combination with the same anchored primer: AAG CTT TTT TTT TTA and the similar random primer. The sequence of random primer is AAGCTTAGTAGGC and AAGCTTTGGTCGA respectively. In addition, the molecular weight of SA5-2 and SA3 is also very approximate. The two gene fragments will be further analyzed in the succedent tests.更多还原